Amino acid transportation, sensing and signal transduction in the mammary gland: key molecular signalling pathways in the regulation of milk synthesis.

The mammary gland, a unique exocrine organ, is responsible for milk synthesis in mammals. Neonatal growth and health are predominantly determined by quality and quantity of milk production. Amino acids are crucial maternal nutrients that are the building blocks for milk protein and are potential energy sources for neonates. Recent advances made regarding the mammary gland further demonstrate that some functional amino acids also regulate milk protein and fat synthesis through distinct intracellular and extracellular pathways. In the present study, we discuss recent advances in the role of amino acids (especially branched-chain amino acids, methionine, arginine and lysine) in the regulation of milk synthesis. The present review also addresses the crucial questions of how amino acids are transported, sensed and transduced in the mammary gland.

Dietary enzymatically-treated Artemisia annua L. supplementation could alleviate oxidative injury and improve reproductive performance of sows reared under high ambient temperature.

The medicinal plant Artemisia annua L. is well known for its antimalarial compound artemisinin and the antioxidant capacity of its active ingredients. However, low bioavailability of Artemisia annua L. limits its therapeutic potential, fermentation of Artemisia annua L. can improve its bioavailability. This study was aimed to investigate the effects of dietary supplementation of enzymatically-treated Artemisia annua L. (EA) on reproductive performance, antioxidant status, milk composition of heat-stressed sows and intestinal barrier integrity of their preweaning offspring. 135 multiparous sows of average parity 4.65 (Landrace × large white) at day 85 of pregnancy were randomly distributed into 3 treatments. Sows in the control group were housed at control rooms (temperature: 27.12 ± 0.18 °C, temperature-humidity index (THI): 70.90 ± 0.80) and fed the basal diet. Sows in the HS, HS + EA groups were fed the basal diet supplemented with 0 or 1.0 g/kg EA respectively, and reared at heat stress rooms (temperature: 30.11 ± 0.16 °C, THI: 72.70 ± 0.60). Heat stress increased the malondialdehyde (MDA) content, reduced the activities of total antioxidant capacity (T-AOC) and total superoxide dismutase (T-SOD) of sows and piglets, and seriously compromised the antioxidant capacity of the sows and the intestinal integrity of their offspring. However, dietary supplementation of 1.0 g/kg EA reduced the MDA content, increased the activities of T-SOD and T-AOC in serum, colostrum, and milk of heat-stressed sows, and increased colostrum yield and 14-d milk fat content. EA supplementation also increased piglet weaning weight and the activities of T-SOD and T-AOC in serum. In addition, the abundances of intestinal tight junction proteins claudin-1 and occludin were up-regulated in piglets in EA-supplemented group. In conclusion, dietary EA supplementation at 1.0 g/kg can alleviate the oxidative stress in heat-stressed sows, improve the antioxidant capacity in both sows and their offspring, and promote the intestinal barrier integrity in their offspring. EA may be a potent dietary supplement that ameliorates oxidative stress in livestock production by improving the antioxidant capacity.

Novel advances in understanding fatty acid-binding G protein-coupled receptors and their roles in controlling energy balance.

Diabetes, obesity, and other metabolic diseases have been recognized as the main factors that endanger human health worldwide. Most of these metabolic syndromes develop when the energy balance in the body is disrupted. Energy balance depends upon the systemic regulation of food intake, glucose homeostasis, and lipid metabolism. Fatty acid-binding G protein-coupled receptors (GPCRs) are widely expressed in various types of tissues and cells involved in energy homeostasis regulation. In this review, the distribution and biological functions of fatty acid-binding GPCRs are summarized, particularly with respect to the gut, pancreas, and adipose tissue. A systematic understanding of the physiological functions of the fatty acid-binding GPCRs involved in energy homeostasis regulation will help in identifying novel pharmacological targets for metabolic diseases.

Current Evidences and Future Perspectives for AMPK in the Regulation of Milk Production and Mammary Gland Biology.

Activated protein kinase (AMP)-activated protein kinase (AMPK) senses the cellular energy status and coordinates catabolic and anabolic processes. Extensive studies have proposed that AMPK regulates energy homeostasis, cell growth, autophagy, mitochondrial biology and inflammation. The biological functions of AMPK vary in different tissues or organs. As a unique organ that produces milk, the mammary gland has recently attracted substantial research attention. This review discusses how AMPK in the mammary gland is activated by energy deprivation and heat stress via the activation of canonical and non-canonical pathways. In addition, the important downstream targets of AMPK and their functions in the mammary gland, especially during milk synthesis, are summarized in the review.

L-carnitine increases cell proliferation and amino acid transporter expression via the activation of insulin-like growth factor I signaling pathway in rat trophoblast cells.

Early embryo implantation and development is primarily determined by the homeostasis between cellular apoptosis and proliferation as well as placental nutrient transporters. Recent studies showed that L-carnitine enhances female reproductive performance. However, the potential function of L-carnitine on placenta is largely unknown. In our study, primary rat trophoblast cells were separated and cultured for 12 hr in medium containing various concentrations of L-carnitine (0, 1, 10, and 50 mM). Placenta trophoblast cells treated with 50 mM L-carnitine increased the proportion of cells in S phase of the cell cycle (p < .05). In addition, live cell percentage was increased when treated with either 10 mM or 50 mM L-carnitine, which was accompanied with decreased necrotic cells, late apoptotic cells, and early apoptotic cells (p < .05). Compared with the control treatment, the mRNA expression of insulin-like growth factor I (IGF-1) and insulin-like growth factor I receptor (IGF-1R) was higher in rat placenta trophoblasts treated with either 10 mM or 50 mM L-carnitine (p < .05). Similarly, sodium-dependent neutral amino acid transporter (SNAT)-1 and SNAT2 were up-regulated in both mRNA and protein levels when trophoblast cells were treated with 50 mM L-carnitine (p < .05). Inhibiting downstream targets (Akt or ERK signaling pathways) of IGF-1 signaling pathway partially blocked the effect the L-carnitine-induced increase in protein abundances of SNAT1 and SNAT2. Collectively, our data showed protective role of L-carnitine on placenta trophoblast cells through the involvement of IGF-1 signaling pathway.

Combined yeast culture and organic selenium supplementation during late gestation and lactation improve preweaning piglet performance by enhancing the antioxidant capacity and milk content in nutrient-restricted sows.

This study was conducted to investigate the effects of dietary supplementation with yeast culture (YC) and organic selenium (Se) during late gestation and lactation on reproductive performance, milk quality, piglet preweaning performance, antioxidant capacity, and secretion of immunoglobulin in multiparous sows. A total of 160 healthy cross-bred sows (Landrace × Yorkshire, mean parity 4.1 ± 0.3) were randomly assigned to 4 groups as follows: 1) high nutrient (HN), 3,420 kcal/kg digestible energy (DE) and 18.0% crude protein (CP); 2) low nutrient (LN), 3,240 kcal/kg DE and 16.0% CP; 3) LN + YC, LN diet + 10 g/kg YC; 4) LN + YC + Se, LN diet + 10 g/kg YC + organic Se (1 mg/kg Se). Feeding trials of sows started from d 85 of pregnancy to d 35 of lactation. Compared with sows in the LN group, sows fed the LN + YC + Se diet had greater litter weaning weight, average litter gain, and milk fat content (14-d and 25-d milk) (P < 0.05). The content of malonaldehyde (MDA) (colostrum and 14-d milk) was lesser, and the activity of glutathione peroxidase (GSH-Px) (colostrum and 25-d milk) was greater when sows were fed the LN + YC + Se diet, compared with sows fed the LN diet (P < 0.05). Supplementation of YC and organic Se in the nutrient-restricted diet improved sows' reproductive performance and pig weaning body weight by enhancing the antioxidant capacity and fat content in milk.

Excessive BCAA regulates fat metabolism partially through the modification of m6A RNA methylation in weanling piglets.

BACKGROUND: Fat percentage and distribution in pigs are associated with their productive efficiency and meat quality. Dietary branched-chain amino acids (BCAA) regulate fat metabolism in weanling piglets with unknown mechanism. It is reported that N6-methyl-adenosine (m6A) is involved in fat metabolism in mice. The current study was designed to investigate the relationship between dietary branched-chain amino acids and fat metabolism through N6-methyl-adenosine (m6A) in weanling piglets. METHODS: A total of 18 healthy crossbred weaned piglets (Duroc × Landrace × Large White, 10.45 ± 0.41 kg) were divided into 3 treatments and were fed the low BCAA dose diet (L-BCAA), the normal dose BCAA diet (N-BCAA), or the high dose BCAA (H-BCAA) diet for 3 weeks. RESULTS: Our results show that compared with the N-BCAA group, the L-BCAA group had higher concentration of serum leptin (P < 0.05), while the H-BCAA group had lower concentration of serum adiponectin (P < 0.05). Fatty acid synthesis in pigs from the H-BCAA group was lower than those from the N-BCAA group with the down-regulation of lipogenic genes (ACACA, FASN, PPAR-r, SREBP-1c in ventral and dorsal fat, SREBP-1c in liver) and up-regulation of lipolysis genes (HSL, ATGL, CPT-1A, FABP4 in ventral fat, HSL in liver) (P < 0.05). Similarly, fatty acid synthesis in pigs from the L-BCAA group was also lower than those from the N-BCAA group with the decrease of lipogenic genes (ACACA in ventral, ACACA and FASN in dorsal fat, ACACA, FASN, SREBP-1c in liver) and the increase of lipolysis genes (ATGL, CPT-1A CD36, FABP4 in ventral fat and HSL, ATGL, CPT-1A in dorsal fat, CPT-1A) (P < 0.05). Feeding H-BCAA diet significantly reduced total m6A levels in ventral and dorsal fat and liver tissues (P < 0.05). The decrease of total m6A is associated with down-regulation of METTL3, METTL14 and FTO in dorsal fat and METTL3 and FTO in liver (P < 0.05). Decreased m6A modification of ACACA and FASN in ventral and dorsal adipose tissues was observed in pig fed with excessive BCAA. CONCLUSION: These results suggest that insufficient or excessive BCAA decreased the fat deposition by increasing lipolysis and deceasing lipogenesis in adipose and liver tissues. Dietary excessive BCAA might regulate the process of lipid metabolism partly through the m6A RNA methylation.

Maternal heat stress regulates the early fat deposition partly through modification of m6A RNA methylation in neonatal piglets.

It is known that heat stress induces various physiological challenges in livestock production including changes in lipid metabolism. However, the molecular mechanism of how heat stress regulates lipid metabolism at the mRNA level is still largely unknown. N6-methyl-adenosine (m6A) is the most common and abundant modification on RNA molecules present in eukaryotes, which affects almost all aspects of RNA metabolism and thus gives us the hint that it may participate in changes of gene expression of lipid metabolism during heat stress. Therefore, the purpose of the present study was to investigate the effect of heat stress on fat metabolism in 21-day Large White × Landrace piglets from sows challenged by heat stress from day 85 of gestation until day 21 of lactation. We measured the expression of heat shock proteins (HSPs), genes associated with lipid metabolism, m6A-related enzymes, and m6A levels in abdominal fat and liver of offspring piglets. Our results showed that high ambient temperature significantly increased the expression of HSP70 (P < 0.01) in both liver and abdominal fat and upregulated HSP27 in the liver (P < 0.05). Additionally, genes involved in fat metabolism such as ACACA, FASN, DGAT1, PPAR-γ, SREBP-1c, and FABP4 were upregulated in abdominal fat in the experimental group challenged by high ambient temperature. In the liver, heat stress increased the mRNA expression of DGAT1, SREBP-1c, and CD36 and decreased ATGL and CPT1A expression (P < 0.05). The m6A level was higher in the heat stress group compared with the control group in the liver and abdominal fat of offspring piglets (P < 0.01). Notably, heat stress also increased gene expression of METTL14, WTAP, FTO, and YTHDF2 (P < 0.05) in both abdominal fat and liver. The protein abundances of METTL3, METTL14, and FTO were upregulated after heat stress in abdominal fat (P < 0.05) but not in the liver. Although there was no difference in the protein abundance of YTHDF2 in abdominal fat, its level was increased in the liver (P < 0.05). In conclusion, our findings showed that heat stress increased expression of genes involved in lipogenesis, which provided scientific evidence to the observation of increased fatness in pigs under heat stress. We also demonstrated a possible mechanism that m6A RNA modification may be associated with these changes in lipid metabolism upon heat stress.

Branched chain amino acids stimulate gut satiety hormone cholecystokinin secretion through activation of the umami taste receptor T1R1/T1R3 using an in vitro porcine jejunum model.

Branched chain amino acids (BCAAs) are essential amino acids involved in regulation of feed intake. The function of BCAAs on the central nervous system has been extensively studied, but effects of BCAAs on secretion of gut satiety hormones and their underlying mechanisms are largely unknown. In this study, we evaluated the distribution of gut hormones and amino acid receptors in the porcine GI tract and found cholecystokinin (CCK) and taste dimeric receptor type 1 member 1/3 (T1R1/T1R3) were predominantly expressed in the jejunum and functionally interrelated. We further evaluated the effects of l-leucine, l-isoleucine, l-valine, and BCAAs on CCK and T1R1/T1R3 expression in porcine jejunum tissue. Our data demonstrated that stimulation of porcine jejunum tissue with 10 mM l-leucine, l-isoleucine or BCAAs mix (l-leucine : l-isoleucine : l-valine = 1 : 0.51 : 0.63) for 2 hours significantly increased mRNA expression and protein abundance of T1R1/T1R3 and secretion of CCK (P < 0.05). However, the l-valine treatment only increased the mRNA and protein abundance of T1R1 and T1R3 (P < 0.05), but not CCK secretion (P > 0.10). l-Leucine-, l-isoleucine- or BCAAs mix-induced CCK secretion was significantly decreased after tissues were pretreated with lactisole, a T1R1/T1R3 inhibitor (P < 0.05). Furthermore, the increased mRNA and protein abundance of T1R1/T1R3 were also largely attenuated by blocking T1R1/T1R3 with lactisole (P < 0.05). l-Leucine, l-isoleucine and BCAAs mix appeared to induce the gut satiety hormone CCK secretion through jejunal T1R1/T1R3. These results indicate over-supplementation with BCAAs in the diet might decrease food intake in swine and humans through gastrointestinal feedback.

Role of Maternal Dietary Protein and Amino Acids on Fetal Programming, Early Neonatal Development, and Lactation in Swine.

Maternal nutrition plays a vital role in fetal development, early development of neonates, and lactation and regulates the lifetime productivity of offspring. During pregnancy, maternal nutrition alters expression of the fetal genome and the development of tissues and organs via fetal programming. After parturition, maternal nutrition continues to regulate growth and development of piglets through maternal milk, which contains carbohydrates, lipids, proteins and oligosaccharides. Thus, deficiencies in maternal nutrition are detrimental to development of piglets, which can lead to inefficient growth and decreased carcass merit. Protein is an important nutritional component for sows, which not only functions in muscle development, but also plays a vital role in embryonic and neonatal development and lactation. Although effects of maternal undernutrition on neonatal development have been widely studied in sows, the function of different maternal dietary protein levels on fetal development, neonatal growth and lactation performance of sows is largely unknown. Determination of the effects and underlying mechanisms of maternal dietary protein levels on development of piglets is vital to the pork industry. Therefore, we summarized recent reports regarding mechanisms of effects of maternal protein levels on regulation of conceptus growth and early postnatal development though uterine fetal programming and lactation in swine.

Dietary Branched-Chain Amino Acids Regulate Food Intake Partly through Intestinal and Hypothalamic Amino Acid Receptors in Piglets.

Strategies to increase feed intake are of great importance for producing more meat in swine production. Intestinal and hypothalamic amino acid receptors are found to largely participate in feed intake regulation. The purpose of the current research is to study the function of branched-chain amino acid (BCAA) supplementation in the regulation of feed intake through sensors that can detect amino acids in piglets. Twenty-four piglets were assigned one of four treatments and fed one of the experimental diets for either a short period (Expt. 1) or a long period (Expt. 2): a normal protein diet (NP, 20.04% CP), a reduced-protein diet (RP, 17.05% CP), or a reduced-protein test diet supplemented with one of two doses of BCAAs (BCAA1, supplemented with 0.13% l-isoleucine, 0.09% l-leucine, and 0.23% l-valine; BCAA2, supplemented with the 150% standardized ileal digestibility BCAA requirement, as recommended by the National Research Council (2012)). In Expt. 1, no differences were observed in the feed intake among piglets fed different diets ( P > 0.05). In Expt. 2, compared with the RP group, the feed intake of piglets was significantly increased after sufficient BCAAs were supplemented in the BCAA1 group, which was associated with decreased cholecystokinin secretion ( P < 0.05), down-regulated expression of type-1 taste receptors 1/3 (T1R1/T1R3) in the intestine, as well as increased expression of pro-opiomelanocortin, activated general control nonderepressible 2 (GCN2), and eukaryotic initiation factor 2α (eIF2α) in the hypothalamus ( P < 0.05). However, the feed intake was decreased for unknown reasons when the piglets were fed a BCAA over-supplemented diet. Our study confirmed that a BCAA-deficient diet inhibited feed intake through two potential ways: regulating the amino acid T1R1/T1R3 receptor in the intestine or activating GCN2/eIF2α pathways in the hypothalamus.

Recent progress of porcine milk components and mammary gland function.

As the only nutritional source for newborn piglets, porcine colostrum and milk contain critical nutritional and immunological components including carbohydrates, lipids, and proteins (immunoglobulins). However, porcine milk composition is more complex than these three components. Recently, scientists identified additional and novel components of sow colostrum and milk, including exosomes, oligosaccharides, and bacteria, which possibly act as biological signals and modulate the intestinal environment and immune status in piglets and later in life. Evaluation of these nutritional and non-nutritional components in porcine milk will help better understand the nutritional and biological function of porcine colostrum and milk. Furthermore, some important functions of the porcine mammary gland have been reported in recent published literature. These preliminary studies hypothesized how glucose, amino acids, and fatty acids are transported from maternal blood to the porcine mammary gland for milk synthesis. Therefore, we summarized recent reports on sow milk composition and porcine mammary gland function in this review, with particular emphasis on macronutrient transfer and synthesis mechanisms, which might offer a possible approach for regulation of milk synthesis in the future.